Rice cultivar M-206

ABSTRACT

A novel rice cultivar, designated M-206, is disclosed. The invention relates to the seeds of rice cultivar M-206, to the plants of rice M-206 and to methods for producing a rice plant produced by crossing the cultivar M-206 with itself or another rice variety. The invention further relates to hybrid rice seeds and plants produced by crossing the cultivar M-206 with another rice cultivar.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive rice cultivar,designated M-206. Rice is an ancient agricultural crop and is today oneof the principal food crops of the world. There are two cultivatedspecies of rice: Oryza sativa L., the Asian rice, and O. glaberrimaSteud., the African rice. O. sativa L. constitutes virtually all of theworld's cultivated rice and is the species grown in the United States.Three major rice producing regions exist in the United States: theMississippi Delta (Arkansas, Mississippi, northeast Louisiana, southeastMissouri), the Gulf Coast (southwest Louisiana, southeast Texas), andthe Central Valleys of California.

Rice is a semiaquatic crop that benefits from flooded soil conditionsduring part or all of the growing season. In the United States, rice isgrown on flooded soils to optimize grain yields. Heavy clay soils orsilt loam soils with hard pan layers about 30 cm below the surface aretypical rice-producing soils because they minimize water losses fromsoil percolation. Rice production in the United States can be broadlycategorized as either dry-seeded or water-seeded. In the dry-seededsystem, rice is sown into a well-prepared seed bed with a grain drill orby broadcasting the seed and incorporating it with a disk or harrow.Moisture for seed germination is from irrigation or rainfall. Anothermethod of planting by the dry-seeded system is to broadcast the seed byairplane into a flooded field, then promptly drain the water from thefield. For the dry-seeded system, when the plants have reachedsufficient size (four- to five-leaf stage), a shallow permanent flood ofwater 5 to 16 cm deep is applied to the field for the remainder of thecrop season.

In the water-seeded system, rice seed is soaked for 12 to 36 hours toinitiate germination, and the seed is broadcast by airplane into aflooded field. The seedlings emerge through a shallow flood, or thewater may be drained from the field for a short period of time toenhance seedling establishment. A shallow flood is maintained until therice approaches maturity. For both the dry-seeded and water-seededproduction systems, the fields are drained when the crop is mature, andthe rice is harvested 2 to 3 weeks later with large combines. In ricebreeding programs, breeders try to employ the production systemspredominant in their respective region. Thus, a drill-seeded breedingnursery is used by breeders in a region where rice is drill-seeded and awater-seeded nursery is used in regions where water-seeding isimportant.

Rice in the United States is classified into three primary market typesby grain size, shape, and chemical composition of the endosperm:long-grain, medium grain and short-grain. Typical U.S. long-graincultivars cook dry and fluffy when steamed or boiled, whereas medium-and short-grain cultivars cook moist and sticky. Long-grain cultivarshave been traditionally grown in the southern states and generallyreceive higher market prices.

Although specific breeding objectives vary somewhat in the differentregions, increasing yield is a primary objective in all programs. Grainyield of rice is determined by the number of panicles per unit area, thenumber of fertile florets per panicle, and grain weight per floret.Increases in any or all of these yield components may provide amechanism to obtain higher yields. Heritable variation exists for all ofthese components, and breeders may directly or indirectly select forincreases in any of them.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety animproved combination of desirable traits from the parental germplasm.These important traits may include higher seed yield, resistance todiseases and insects, better stems and roots, tolerance to lowtemperatures, and better agronomic characteristics on grain quality.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection, ora combination of these methods.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three or more years. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits maybe used as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to 12 years from the time thefirst cross is made and may rely on the development of improved breedinglines as precursors. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of plant breeding is to develop new, unique and superior ricecultivars and hybrids. The breeder initially selects and crosses two ormore parental lines, followed by self pollination and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing and mutations. The breeder has no direct control at the cellularlevel. Therefore, two breeders will never develop the same line, or evenvery similar lines, having the same rice traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same cultivar twice by using theexact same original parents and the same selection techniques. Thisunpredictability results in the expenditure of large amounts of researchmonies to develop superior new rice cultivars.

The development of new rice cultivars requires the development andselection of rice varieties, the crossing of these varieties andselection of superior hybrid crosses. The hybrid seed is produced bymanual crosses between selected male-fertile parents or by using malesterility systems. These hybrids are selected for certain single genetraits such as semidwarf plant type, pubescence, awns, and apiculuscolor which indicate that the seed is truly a hybrid. Additional data onparental lines, as well as the phenotype of the hybrid, influence thebreeder's decision whether to continue with the specific hybrid cross.

Pedigree breeding and recurrent selection breeding methods are used todevelop cultivars from breeding populations. Breeding programs combinedesirable traits from two or more cultivars or various broad-basedsources into breeding pools from which cultivars are developed byselfing and selection of desired phenotypes. The new cultivars areevaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents which possess favorable,complementary traits are crossed to produce an F₁. An F₂ population isproduced by selfing one or several F₁'s. Selection of the bestindividuals may begin in the F₂ population; then, beginning in the F₃,the best individuals in the best families are selected. Replicatedtesting of families can begin in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, rice breeders commonly harvest one or moreseeds from each plant in a population and thresh them together to form abulk. Part of the bulk is used to plant the next generation and part isput in reserve. The procedure has been referred to as modifiedsingle-seed descent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh panicles with a machine than to removeone seed from each by hand for the single-seed procedure. Themultiple-seed procedure also makes it possible to plant the same numberof seeds of a population each generation of inbreeding. Enough seeds areharvested to make up for those plants that did not germinate or produceseed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Rice, Oryza sativa L., is an important and valuable field crop. Thus, acontinuing goal of plant breeders is to develop stable, high yieldingrice cultivars that are agronomically sound. The reasons for this goalare obviously to maximize the amount of grain produced on the land usedand to supply food for both animals and humans. To accomplish this goal,the rice breeder must select and develop rice plants that have thetraits that result in superior cultivars.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel rice cultivar,designated M-206. This invention thus relates to the seeds of ricecultivar M-206, to the plants of rice M-206 and to methods for producinga rice plant produced by crossing the rice M-206 with itself or anotherrice line.

Thus, any such methods using the rice variety M-206 are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using rice variety M-206as a parent are within the scope of this invention. Advantageously, therice variety could be used in crosses with other, different, rice plantsto produce first generation (F₁) rice hybrid seeds and plants withsuperior characteristics.

In another aspect, the present invention provides for single geneconverted plants of M-206. The single transferred gene may preferably bea dominant or recessive allele. Preferably, the single transferred genewill confer such traits as herbicide resistance, insect resistance,resistance for bacterial, fungal, or viral disease, male fertility, malesterility, enhanced nutritional quality, and industrial usage. Thesingle gene may be a naturally occurring rice gene or a transgeneintroduced through genetic engineering techniques.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of rice plant M-206. The tissue culture willpreferably be capable of regenerating plants having the physiologicaland morphological characteristics of the foregoing rice plant, and ofregenerating plants having substantially the same genotype as theforegoing rice plant. Preferably, the regenerable cells in such tissuecultures will be embryos, protoplasts, meristematic cells, callus,pollen, leaves, anthers, root tips, flowers, seeds, panicles or stems.Still further, the present invention provides rice plants regeneratedfrom the tissue cultures of the invention.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Days to 50% heading. Average number of days from seeding to the day when50% of all panicles are exerted at least partially through the leafsheath. A measure of maturity.

Grain Yield. Grain yield is measured in pounds per acre and at 14.0%moisture. Grain yield of rice is determined by the number of paniclesper unit area, the number of fertile florets per panicle, and grainweight per floret.

Lodging Percent. Lodging is measured as a subjective rating and ispercentage of the plant stems leaning or fallen completely to the groundbefore harvest.

Grain Length (L). Length of a rice grain is measured in millimeters.

Grain Width (W). Width of a rice grain is measured in millimeters.

Length/Width (L/W) Ratio. This ratio is determined by dividing theaverage length (L) by the average width (W).

1000 Grain Wt. The weight of 1000 rice grains as measured in grams.

Harvest Moisture. The percent of moisture of the grain when harvested.

Plant Height. Plant height in centimeters is taken from soil surface tothe tip of the extended panicle at harvest.

Apparent Amylose Percent. The most important grain characteristic thatdescribes cooking behavior in each grain class, or type, i.e., long,medium, and short grain. The percentage of the endosperm starch ofmilled rice that is amylose. Standard long grains contain 20 to 23%amylose. Rexmont type long grains contain 24 to 25% amylose. Short andmedium grains contain 16 to 19% amylose. Waxy rice contains 0% amylose.Amylose values will vary over environments.

Alkali Spreading Value Indicator of gelatinization temperature and anindex that measures the extent of disintegration of milled rice kernelin contact with dilute alkali solution. Standard medium grains have 6 to7 Alkali Spreading Value (intermediate gelatinization temperature).

RVA Viscosity. Rapid Visco Analyzer is a new and widely used laboratoryinstrument to examine paste viscosity, or thickening ability of milledrice during the cooking process.

Hot Paste Viscosity. Viscosity measure of rice flour/water slurry afterbeing heated to 95° C. Lower values indicate softer and more stickycooking types of rice.

Cool Paste Viscosity. Viscosity measure of rice flour/water slurry afterbeing heated to 95° C. and uniformly cooled to 50° C. (AmericanAssociation of Cereal Chemist). Values less than 200 for cool pasteindicate softer cooking types of rice.

Allele. Allele is any of one or more alternative forms of a gene, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics, except for the characteristics derived from theconverted gene.

Quantitative Trait Loci (QTL). Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Single Gene Converted (Conversion). Single gene converted (conversion)plant refers to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a variety are recovered in additionto the single gene transferred into the variety via the backcrossingtechnique or via genetic engineering.

DETAILED DESCRIPTION OF THE INVENTION

M-206 is a very early to early, semidwarf, medium-grain experimentalline that has been evaluated in large plot yield tests since 1998. Itwas tested under the experimental designation 98-Y-242. It is an earlyselection from the 1991-1992 winter greenhouse cross R18087 and has thepedigree S-301/M204. M-206 was developed by the California CooperativeRice Research Foundation (CCRRF) at the Rice Experiment Station (RES),Biggs, Calif. Both cultivars used in the pedigree were developed by theCalifornia Cooperative Rice Research Foundation, (CCRRF) at the RiceExperiment Station (RES), Biggs, Calif. S-301 is an intermediatematurity short-grain semidwarf that is no longer in production. M-204 isan early maturing Calrose medium-grain with semidwarf height. M-206 isthe product of modified pedigree breeding. Early spray painted panicleselections from the 1993 F₂ nursery selected for medium grain shape werebulked 6 panicles per row in the 1993-94 Hawaiian nursery. Panicleselections grouped from the R18087 Hawaii rows were planted in 1994individual rows. The line of descent from was 94 row 63811 viaindividual panicle selection to 95 row 6309 to 96 row 5136 which wasbulked for 97 small plot 351 that was the seed source for entry 98-Y-242in the RES Preliminary Yield T est. It has been tested in the Universityof California Cooperative Extension (UCCE) Statewide Yield Tests(99-Y-84, 00-Y-63, 01-Y-12, 01-Y-72, 02-Y-11, 02-Y-73) 1999 to 2002.

Panicle selections were first taken for headrow production in 1999 andcontinued through 2002. The 1999 panicles were initially purified in the2000 headrow nursery. Year 2000 selected headrows were generationadvanced in 2000-01 Hawaiian nursery. Year 2001 breeder headrows totaled300 rows from 2000 selected headrows and their descendents from 2001-02Hawaii nursery. The year 2001 breeder increase consisted of twocomponents. One component was derived from bulked year 2000 headrowswhile the other came from the 2000-01 Hawaii nursery. These bulks wereharvested separately and planted in different foundation field locationsin 2002 for a total of 7.7 acres. Previous experience revealed that incertain years more outcrossing occurs in the Hawaii nursery resulting inmore offtypes in either the breeder and/or foundation production fields.Two years of observations and plant counts demonstrated that 2000-01outcrossing in Hawaii was near non-existent resulting in the twocomponents being combined for the planting and production of 2002Foundation seed. The 2001 headrow panicle selections were used to plant400 headrows in 2002.

During the headrow production any putative segregating or variant rowswere removed from the field. Visual inspection (seedling vigor, headingdate, plant height, grain shape and size, etc.) of headrows were used ascriteria to confirm cultivar purity. This is standard procedure at RESto monitor and maintain breeder seed of the 13 currently grownCalifornia rice varieties. M-206 was approved for certification by theCalifornia Crop Improvement Association in 2003. Classes of seed will bebreeder, foundation, registered, and certified seed produced inCalifornia. Foundation seed can be used to produce foundation seed ifnecessary and headrow and breeder seed will be produced in foundationfields as necessary to maintain cultivar purity.

M-206 has been observed in seed increase and production fields for fivegenerations (1999 to 2002) and found to be uniform and stable, asdescribed in the following variety description information. It has beenincreased with continued observation for uniformity.

Rice cultivar M-206 has the following morphologic and othercharacteristics (based primarily on data collected at Biggs, Calif.).

Variety Description Information Maturity (Biggs, Calif. at 135 kg N/ha)

Days to maturity: 79

5 days earlier than M-202

Culm (Degrees from Perpendicular After Flowering)

Angle: Erect (less than 30°)

Length: 96.6 cm (Soil level to top of extended panicle on main stem)

Shorter than M-202 by 3.0 cm

Height Class: Semi-dwarf

Internode Color (After flowering): Green

Strength (Lodging resistance): Moderately strong (most plants leaning)

Flag Leaf (After Heading)

Length: 39.4 cm

Width: 12.2 mm

Pubescence: Glabrous

Leaf Angle (after heading): Erect

Blade Color: Green

Basal Leaf Sheath Color: Green

Ligule

Color (Late vegetative state): White

Shape: Acute to acuminate

Collar Color_((Late vegetative stage)): Pale green

Auricle Color_((Late vegetative stage)): Pale green

Panicle

Length: 19.8 cm

Type: Intermediate

Secondary Branching: Light

Exsertion_((near maturity)): 100%

Axis: Droopy

Shattering: Very low (1%)

Threshability: Easy

Grain (Spikelet)

Awns_((After full heading)): Short and partly awned

Apiculus Color_((At maturity)): Straw

Stigma Color: White

Lemma and Palea Color_((At maturity)): Straw

Lemma and Palea Pubescence: Glabrous, with hair on lemma keel

Spikelet Sterility_((At maturity)): Highly fertile (>90%)

Grain (Seed)

Seed Coat Color: Light brown

Endosperm Type: Nonglutinous (nonwaxy)

Endosperm Translucency: Clear

Endosperm Chalkiness: Small (less than 10% of sample)

Scent: Nonscented

Shape Class (Length/width ratio):

-   -   Paddy—Medium (2.3:1 to 3.3:1)    -   Brown—Medium (2.1:1 to 3.0:1)    -   Milled—Medium (2.0:1 to 2.9:1)

Measurements: (2001-2002 average) Length Width L/W 1000 Grains (mm) (mm)Ratio (grams) Paddy 8.46 3.15 2.7 29.4 Brown 6.20 2.78 2.3 24.0 Milled5.84 2.71 2.2 20.7

Milling Yield_((% whole kernel (head) rice to rough rice)): 66.0

Brown Rice Protein (Leco): 6.9%

Apparent Amylose: 18.0%

Alkali Spreading value: 6.0₍1.5% KOH Solution) 6.4% (1.7% KOH Solution)

Gelatinization Temperature (Differential Scanning Calorimetry) 68.4° C.

Gelatinization Temperature Type: Low

Amylographic Paste Viscosity (Rapid Visco Amylograph AACC Method - RVU)Peak 288 Hot Paste 150 Cooled Paste 253 Consistency 104 Breakdown 139Pasting Temperature:  71.5° C.

Resistance to Low Temperature

Germination and Seedling Vigor: High

Flowering_((Spikelet fertility)): High

Seedling Vigor Not Related to Low Temperature

Vigor: High

Disease Resistance

Aggregate Sheath Spot Rhizoctonia oryzae-sativae: Mod. Susceptible

Stem Rot Sclerotium oryzae: Mod. Susceptible

Blast Resistance Pyrisclaria oryzae race IG1: Susceptible

Insect Resistance

Rice Water Weevil Lissorhoptrus oryzophilus: Susceptible

This invention is also directed to methods for producing a rice plant bycrossing a first parent rice plant with a second parent rice plant,wherein the first or second rice plant is the rice plant from the lineM-206. Further, both first and second parent rice plants may be from thecultivar M-206. Therefore, any methods using the cultivar M-206 are partof this invention: selfing, backcrosses, hybrid breeding, and crosses topopulations. Any plants produced using cultivar M-206 as a parent arewithin the scope of this invention.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cells of tissue culture from which rice plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as pollen, flowers, embryos, ovules,seeds, pods, leaves, stems, anthers and the like. Thus, another aspectof this invention is to provide for cells which upon growth anddifferentiation produce a cultivar having essentially all of thephysiological and morphological characteristics of M-104.

Culture for expressing desired structural genes and cultured cells areknown in the art. Also as known in the art, rices are transformable andregenerable such that whole plants containing and expressing desiredgenes under regulatory control may be obtained. General descriptions ofplant expression vectors and reporter genes and transformation protocolscan be found in Gruber, et al., “Vectors for Plant Transformation, inMethods in Plant Molecular Biology & Biotechnology” in Glich, et al.,(Eds. pp. 89-119, CRC Press, 1993). Moreover GUS expression vectors andGUS gene cassettes a re available from Clone Tech Laboratories, Inc.,Palo Alto, Calif. while luciferase expression vectors and luciferasegene cassettes are available from Pro Mega Corp. (Madison, Wis.).General methods of culturing plant tissues are provided for example byMaki, et al., “Procedures for Introducing Foreign DNA into Plants” inMethods in Plant Molecular Biology & Biotechnology, Glich, et al., (Eds.pp. 67-88 CRC Press, 1993); and by Phillips, et al., “Cell-TissueCulture and In-Vitro Manipulation” in Corn & Corn Improvement, 3rdEdition; Sprague, et al., (Eds. pp. 345-387) American Society ofAgronomy Inc., 1988. Methods of introducing expression vectors intoplant tissue include the direct infection or co-cultivation of plantcells with Agrobacterium tumefaciens, Horsch et al., Science, 227:1229(1985). Descriptions of Agrobacterium vectors systems and methods forAgrobacterium-mediated gene transfer provided by Gruber, et al., supra.

Useful methods include but are not limited to expression vectorsintroduced into plant tissues using a direct gene transfer method suchas microprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably expression vectors are introduced into planttissues using the microprojectile media delivery with the biolisticdevice Agrobacterium-medicated transformation. Transformant plantsobtained with the protoplasm of the invention are intended to be withinthe scope of this invention.

The present invention contemplates a rice plant regenerated from atissue culture of a variety (e.g., M-206) or hybrid plant of the presentinvention. As is well known in the art, tissue culture of rice can beused for the in vitro regeneration of a rice plant. Tissue culture ofvarious tissues of rices and regeneration of plants therefrom is wellknown and widely published. For example, reference may be had to Chu, Q.R., et al., (1999) “Use of bridging parents with high antherculturability to improve plant regeneration and breeding value in rice”,Rice Biotechnology Quarterly 38:25-26; Chu, Q. R., et al., (1998), “Anovel plant regeneration medium for rice anther culture of Southern U.S.crosses”, Rice Biotechnology Quarterly 35:15-16; Chu, Q. R., et al.,(1997), “A novel basal medium for embryogenic callus induction ofSouther US crosses”, Rice Biotechnology Quarterly 32:19-20; and Oono,K., “Broadening the Genetic Variability By Tissue Culture Methods”, Jap.J. Breed. 33 (Suppl.2), 306-307, illus. 1983, the disclosures of whichare hereby incorporated herein in their entirety by reference. Thus,another aspect of this invention is to provide cells which upon growthand differentiation produce rice plants having the physiological andmorphological characteristics of variety M-206.

TABLE 1 Agronomic characteristics and performance of M-206 and M-202 -1998 to 2002 UCCE Statewide Yield Tests Character M-206 M-202 Seedlingvigor (score)†   4.5   4.6 Days 50% heading  85*  89 Plant height (cm) 95  97 Lodging (%)  31  38 Blanking - Greenhouse (%)  12  25 Blanking -Davis (%)  12  16 Blanking - San Joaquin (%)   7  27 Overall blankingmean (%)  10  23 Stem rot (score)   5.4   5.5 Harvest moisture (%)  19.8 19.7 Yield (lb/acre @ 14%) 9350* 9060 †Seedling vigor visual scorewhere 1 = poor and 5 = excellent. *Significantly different at the 0.05probability level.

TABLE 2 Milling yields of M-202 and M-206 - 1998 to 2002 Character M-206M-202 Total milled rice (%) 69.0 68.7 Whole kernel milled rice (%) 66.064.2 Number of samples† 51 30 †Moisture samples outside the 17-25% rangewere removed from the calculation.

TABLE 3 Agronomic Characteristics for M-206 and M-202 at cold location(UCCE @ San Joaquin County) 2001-2002 Character M-206 M-202 Seedlingvigor (score)   4.6   4.7 Days 50% heading  97  98 Plant height (cm)  86 83 Lodging (%)   1   1 Harvest moisture (%)  23.9  24.0 Yield (lb/acre@ 14%) 8590* 7880 †Seedling vigor visual score where 1 = poor and 5 =excellent. *Significantly different at the 0.05 probability level.

When the term rice plant is used in the context of the presentinvention, this also includes any single gene conversions of thatvariety. The term single gene converted plant as used herein refers tothose rice plants which are developed by a plant breeding techniquecalled backcrossing or via genetic engineering techniques whereinessentially all of the desired morphological and physiologicalcharacteristics of a variety are recovered in addition to the singlegene transferred into the variety via the backcrossing technique or viagenetic engineering. Backcrossing methods can be used with the presentinvention to improve or introduce a characteristic into the variety. Theterm backcrossing as used herein refers to the repeated crossing of ahybrid progeny back to the recurrent parent. The parental rice plantwhich contributes the gene for the desired characteristic is termed thenonrecurrent or donor parent. This terminology refers to the fact thatthe nonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental rice plant to which the gene orgenes from the nonrecurrent parent are transferred is known as therecurrent parent as it is used for several rounds in the backcrossingprotocol (Poehlman & Sleper, 1994; Fehr, 1987). In a typical backcrossprotocol, the original variety of interest (recurrent parent) is crossedto a second variety (nonrecurrent parent) that carries the single geneof interest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a rice plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single gene of the recurrent variety ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross, one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new variety but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic, examples of these traits include but are not limited to,male sterility, herbicide resistance, resistance for bacterial, fungal,or viral disease, insect resistance, male fertility, enhancednutritional quality, industrial usage, yield stability and yieldenhancement. These genes are generally inherited through the nucleus.

Further Embodiments of the Invention

This invention also is directed to methods for producing a rice plant bycrossing a first parent rice plant with a second parent rice plantwherein either the first or second parent rice plant is a hybrid riceplant M-206. Further, both first and second parent rice plants can comefrom the hybrid rice M-206. Still further, this invention also isdirected to methods for producing a hybrid rice line M-206-derived riceplant by crossing hybrid rice line M-206 with a second rice plant andgrowing the progeny seed, and repeating the crossing and growing stepswith the hybrid rice line M-206-derived plant from 0 to 7 times. Thus,any such methods using the hybrid rice line M-206 are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using hybrid rice lineM-206 as a parent are within the scope of this invention, includingplants derived from hybrid rice line M-206.

It should be understood that the parents of hybrid M-206 can, throughroutine manipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which rice plants can be regenerated,plant calli, plant clumps and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, kernels,panicles, hulls, leaves, glumes, stems, roots, root tips, anthers,pistils, styles and the like.

Duncan, et al., Planta 165:322-332 (1985) reflects that 97% of theplants cultured that produced callus were capable of plant regeneration.Subsequent experiments with both inbreds and hybrids produced 91%regenerable callus that produced plants. In a further study in 1988,Songstad, et al., Plant Cell Reports 7:262-265 (1988), reports severalmedia additions that enhance regenerability of callus of two inbredlines. Other published reports also indicated that “nontraditional”tissues are capable of producing somatic embryogenesis and plantregeneration. K. P. Rao et al., Maize Genetics Cooperation Newsletter,60:64-65 (1986), refers to somatic embryogenesis from glume calluscultures and B. V. Conger, et al., Plant Cell Reports, 6:345-347 (1987)indicates somatic embryogenesis from the tissue cultures of corn leafsegments. Thus, it is clear from the literature that the state of theart is such that these methods of obtaining plants are, and were,“conventional” in the sense that they are routinely used and have a veryhigh rate of success.

Tissue culture of corn is described in European Patent Application,publication 160,390, incorporated herein by reference. Corn tissueculture procedures are also described in Green and Rhodes, “PlantRegeneration in Tissue Culture of Maize,” Maize for Biological Research(Plant Molecular Biology Association, Charlottesville, Va. 367-372,(1982)) and in Duncan et al., “The Production of Callus Capable of PlantRegeneration from Immature Embryos of Numerous Zea Mays Genotypes,” 165Planta 322:332 (1985). Thus, another aspect of this invention is toprovide cells which upon growth and differentiation produce rice plantshaving the physiological and morphological characteristics of hybridrice line M-206.

The utility of hybrid rice line M-206 also extends to crosses with otherspecies. Commonly, suitable species will be of the family Graminaceae,and especially of the genera Zea, Tripsacum, Croix, Schlerachne,Polytoca, Chionachne, and Trilobachne, of the tribe Maydeae. Potentiallysuitable for crosses with M-206 may be the various varieties of grainsorghum, Sorghum bicolor (L.) Moench.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed hybrid.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed rice plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the rice plant(s).

Expression Vectors for Corn Transformation

Marker Genes—Expression vectors include at least one genetic marker,operably linked to a regulatory element (a promoter, for example) thatallows transformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990<Hille et al., Plant Mol. Biol. 7:171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or broxynil. Comai et al., Nature317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) andStalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include -glucuronidase (GUS, -galactosidase,luciferase and chloramphenicol, acetyltransferase. Jefferson, R. A.,Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989),Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987), DeBlock etal., EMBO J. 3:1681 (1984). Another approach to the identification ofrelatively rare transformation events has been use of a gene thatencodes a dominant constitutive regulator of the Zea mays anthocyaninpigmentation pathway. Ludwig et al., Science 247:449 (1990).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes publication 2908, Imagene Green™, p. 1-4 (1993) and Naleway etal., J. Cell Biol. 115:151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters—Genes included in expression vectors must be driven bynucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression inrice. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in rice. With an inducible promoter the rate oftranscription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression inrice or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in rice.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)).

The ALS promoter, Xba1/NcoI fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xba1/NcoIfragment), represents a particularly useful constitutive promoter. SeePCT application WO96/30530.

C. Tissue-specific or Tissue-preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin rice. Optionally, the tissue-specific promoter is operably linked toa nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in rice. Plants transformed with a geneof interest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondroin or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is rice. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RFLP, PCR andSSR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant inbred line can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. Tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of aBt-endotoxin gene. Moreover, DNA molecules encoding -endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

C. A lectin. See, for example, the disclose by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor 1), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper accumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-, lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-1,4-D-polygalacturonases facilitate fungalcolonization and plant nutrient release by solubilizing plant cell wallhomo- -1,4-D-galacturonase. See Lamb et al., Bio/Technology 10:1436(1992). The cloning and characterization of a gene which encodes a beanendopolygalacturonase-inhibiting protein is described by Toubart et al.,Plant J. 2:367 (1992).

R. A development-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bioi/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes that Confer Resistance to a Herbicide, for Example:

A. A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase, bar, genes), and pyridinoxy orphenoxy propionic acids and cycloshexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai.European patent application No. 0 333 033 to Kumada et al., and U.S.Pat. No. 4,975,374 to Goodman et al., disclose nucleotide sequences ofglutamine synthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al., DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy propionic acids and cycloshexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

C. A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

3. Genes that Confer or Contribute to a Value-added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.U.S.A. 89:2624 (1992).

B. Decreased Phytate Content

-   -   1) Introduction of a phytase-encoding gene would enhance        breakdown of phytate, adding more free phosphate to the        transformed plant. For example, see Van Hartingsveldt et al.,        Gene 127:87 (1993), for a disclosure of the nucleotide sequence        of an Aspergillus niger phytase gene.    -   2) A gene could be introduced that reduced phytate content. In        maize, this, for example, could be accomplished, by cloning and        then reintroducing DNA associated with the single allele which        is responsible for maize mutants characterized by low levels of        phytic acid. See Raboy et al., Maydica 35:383 (1990).

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus lichenifonnis-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme 11).

Methods for Corn Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985). A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber et al.,supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer

Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice and corn. Hiei etal., The Plant Journal 6:271-282 (1994) and U.S. Pat. No. 5,591,616issued Jan. 7, 1997. Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 μm. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299(1988), Klein et al., Bio/Technology 6:559-563 (1988), Sanford, J. C.,Physiol Plant 7:206 (1990), Klein et al., Biotechnology 10:268 (1992).In corn, several target tissues can be bombarded with DNA-coatedmicroprojectiles in order to produce transgenic plants, including, forexample, callus (Type I or Type II), immature embryos, and meristematictissue.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-omithine have also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24:51-61 (1994).

Following transformation of rice target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic inbred line. The transgenic inbred line couldthen be crossed, with another (non-transformed or transformed) inbredline, in order to produce a new transgenic inbred line. Alternatively, agenetic trait which has been engineered into a particular rice lineusing the foregoing transformation techniques could be moved intoanother line using traditional backcrossing techniques that are wellknown in the plant breeding arts. For example, a backcrossing approachcould be used to move an engineered trait from a public, non-eliteinbred line into an elite inbred line, or from an inbred line containinga foreign gene in its genome into an inbred line or lines which do notcontain that gene. As used herein, “crossing” can refer to a simple X byY cross, or the process of backcrossing, depending on the context.

When the term inbred rice plant is used in the context of the presentinvention, this also includes any single gene conversions of thatinbred. The term single gene converted plant as used herein refers tothose rice plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of an inbred are recovered in additionto the single gene transferred into the inbred via the backcrossingtechnique. Backcrossing methods can be used with the present inventionto improve or introduce a characteristic into the inbred. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to one of the parental rice plants for that inbred. Theparental rice plant which contributes the gene for the desiredcharacteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental rice plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman & Sleper,1994; Fehr, 1987). In a typical backcross protocol, the original inbredof interest (recurrent parent) is crossed to a second inbred(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a riceplant is obtained wherein essentially all of the desired morphologicaland physiological characteristics of the recurrent parent are recoveredin the converted plant, in addition to the single transferred gene fromthe nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalinbred. To accomplish this, a single gene of the recurrent inbred ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original inbred. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross, one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic, examples of these traits include but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Some known exceptions to this are the genes for male sterility,some of which are inherited cytoplasmically, but still act as singlegene traits. Several of these single gene traits are described in U.S.Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, the disclosures of whichare specifically hereby incorporated by reference.

Deposit Information

A deposit of the California Cooperative Rice Research Foundationproprietary rice cultivar M-206 disclosed above and recited in theappended claims has been made with the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110. The date ofdeposit was Jun. 18, 2003. The deposit of 2,500 seeds were taken fromthe same deposit maintained by the California Cooperative Rice ResearchFoundation since prior to the filing date of this application. Allrestrictions upon the deposit have been removed, and the deposit isintended to meet all of the requirements of 37 C.F.R. §1.801-1.809. TheATCC accession number is PTA-5274. The deposit will be maintained in thedepository for a period of 30 years, or 5 years after the last request,or for the effective life of the patent, whichever is longer, and willbe replaced as necessary during that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

1. A rice seed designated M-206, wherein a representative sample of saidseed has been deposited under ATCC Accession No. PTA-5274.
 2. A riceplant, or a part thereof, produced by growing the seed of claim
 1. 3.Pollen of the plant of claim
 2. 4. An ovule of the plant of claim
 2. 5.A rice plant, or a part thereof, having all of the physiological andmorphological characteristics of the rice plant of claim
 2. 6. A tissueculture of regenerable cells or protoplasts produced from the rice plantof claim
 2. 7. The tissue culture of claim 6 wherein the cells orprotoplasts of the tissue culture are produced from a tissue selectedfrom the group consisting of embryos, meristematic cells, pollen,leaves, anthers, roots, root tips, flowers, seeds, and stems.
 8. A riceplant regenerated from the tissue culture of claim 7, wherein said planthas all the physiological and morphological characteristics of the riceplant grown from rice seed designated M-206.
 9. A method for producing arice seed wherein the method comprises crossing a first parent riceplant with a second parent rice plant and harvesting the resultanthybrid rice seed, wherein said first parent rice plant or second parentrice plant is the rice plant of claim
 2. 10. A method of producing anherbicide resistant rice plant wherein the method comprises transformingthe rice plant of claim 2 with a transgene that confers herbicideresistance.
 11. An herbicide resistant rice plant produced by the methodof claim
 10. 12. A method of producing an insect resistant rice plantwherein the method comprises transforming the rice plant of claim 2 witha transgene that confers insect resistance.
 13. An insect resistant riceplant produced by the method of claim
 12. 14. A method of producing adisease resistant rice plant wherein the method comprises transformingthe rice plant of claim 2 with a transgene that confers diseaseresistance.
 15. A disease resistant rice plant produced by the method ofclaim
 14. 16. A method of producing a rice plant with modified fattyacid or carbohydrate metabolism wherein the method comprisestransforming the rice plant of claim 2 with one or more transgenesencoding a protein selected from the group consisting offructosyltransferase, levansucrase, alpha-amylase, invertase and starchbranching enzyme; or comprising an antisense gene of stearyl-ACPdesaturase.
 17. A rice plant produced by the method of claim
 16. 18. Amethod of introducing a desired trait into rice cultivar M-206 whereinthe method comprises: (a) crossing the M-206 plants, representative seeddeposited under ATCC Accession No. PTA-5274, with plants of another riceline that comprise a desired trait to produce F1 progeny plants, whereinthe desired trait is selected from the group consisting of malesterility, herbicide resistance, insect resistance and resistance tobacterial, fungal or viral disease; (b) selecting F1 progeny plants thathave the desired trait to produce selected F1 progeny plants; (c)crossing the selected F1 progeny plants with the M-206 plants to producefirst backcross progeny plants; (d) selecting for first backcrossprogeny plants that have the desired trait and physiological andmorphological characteristics of rice cultivar M-206 to produce selectedfirst backcross progeny plants; and (e) repeating steps (c) and (d)three or more times in succession to produce selected fourth or higherbackcross progeny plants that comprise the desired trait and all of thephysiological and morphological characteristics of rice cultivar M-206as described in the VARIETY DESCRIPTION INFORMATION and as determined ata 5% significance level when grown in the same environmental conditions.19. A plant produced by the method of claim 18, wherein the plant hasthe desired trait and all of the physiological and morphologicalcharacteristics of rice cultivar M-206 as described in the VARIETYDESCRIPTION INFORMATION and as determined at a 5% significance levelwhen grown in the same environmental conditions.